细胞自噬对酒精诱导的肝细胞毒性的保护作用

Protective Effect of Autophagy on Ethanol-induced Hepatotoxicity

目的:研究细胞自噬对酒精诱导的人肝细胞系(CL-1)的保护作用。方法:培养正常肝细胞系CL-1细胞,80 mmol/L酒精常规处理24小时,采用CCK-8法观察酒精对细胞活力的影响;流式细胞技术观察酒精对细胞凋亡的影响;免疫蛋白印迹及转染GFP-LC3法检测细胞自噬水平;选用rapamycin和3-MA调节细胞自噬,观察酒精处理后细胞活力及凋亡的变化。结果:酒精处理体外培养的CL-1细胞,实验组较对照组细胞活力下降(P<0.05);实验组细胞46.2%发生凋亡,显著高于对照组8.4%;LC3Ⅱ及Beclin1水平显著高于对照组;GFP-LC3荧光数显著高于对照组(P<0.05);调节细胞自噬水平,rapamycin组细胞活性增加(P<0.01),31.1%(46.2%)细胞发生凋亡;3-MA组细胞活性降低(P<0.05),54.1%(46.2%)细胞发生凋亡。结论:酒精处理降低CL-1细胞活性,促进凋亡,提高自噬水平;提高或降低细胞自噬水平,细胞凋亡及活力随之降低和增加;细胞自噬能够对抗酒精诱导的肝细胞凋亡。

Objective： To investigate the effect of autophagy on ethanol-induced human hepatocyte line （CL-1）. Methods： CL-1 cells were cultured under the conventional condition and treated with 80 mmol/L ethanol for 24 hours in order to observe ethanol-induced cell viability by flow cytometry and CCK-8; Westen blot and transient GFP-LC3 were performed to analyze the level of autophagy. Meanwhile, CL-1 cells were pretreated with rapamycin and 3-MA to detect cell viability and apoptosis. Results： After treated with ethanol, cell viability was decreased （P〈0.05） while the level of apoptosis and autophagy were increased. Level of apoptosis was 46.2% in alcohol-treated group versus 8.4% in control group. LC3 II and Beclin 1 were significantly higher than that in the control group. The number of GFP-LC3 fluorescent particles was significantly higher than that in control group （P〈0.05）. Correspondingly, after pretreatment, cell activity in rapamycin group increased （P〈0.01） and the level of apoptosis decreased from 46.2 % to 31.1%, while cell activity in 3-MA group decreased （P〈0.05） and the level of apoptosis increased from 46.2 % to 54.1%. Conclusion： Alcohol treatment reduces CL-1 cell activity, promotes apoptosis, and improves the level of autophagy. Regulating the level of autophagy with either improvement or reduction markedly reduced or improved the cell viability and apoptosis. Autophagy protects against ethanol-induced apoptosis in hepatocyte.